Smallpox virus and/or genetically-engineered orthopoxvimses are considered one of the most significant threats for malevolent use as potential agents of bioterrorism. Because smallpox was eliminated from the U.S. population in the 1960's, prophylactic immunization was discontinued. The subsequent 40 years have produced a population that is immunologically naive and highly susceptible to orthopoxvirus infection. Due to the small but significant risk of serious complications from vaccination, mass immunization of the populace is contra-indicated. 'Therefore, the focus of the experiments outlined in this proposal is to develop an effective anti-poxvirus drug for use in treating or preventing human disease caused by pathogenic poxviruses. The target of our antiviral drug development efforts will be the poxvirus proteinase responsible for core protein maturation, a step which is absolutely essential for the production and spread of infectious virions. This project will be carried out as a partnership between an academic group at Oregon State University with a long history of research in various aspects of poxvirus proteolysis, and a biopharmaceutical company, SIGA Research Laboratories, which is actively engaged in the development of proteinase inhibitors as anti- infectives. Together, these groups will identify' the viral gene product responsible for catalyzing core protein maturation and use genetic approaches to validate it as an antiviral target. Expression vector technology will be used to express and purify the large quantities of the core protein proteinase. The purified proteinase will serve as the starting material for a two-pronged approach to the identification of potential inhibitors: l) Structure-function analysis coupled with rational drug design; and 2) Development of an in vitro cleavage assay appropriate for use in high-throughput screening against limited libraries of potential proteinase inhibitors. Lead compounds identified by either approach will be tested for the ability to inhibit the replication of various orthopoxviruses in tissue culture cells. If necessary, lead compounds will be subjected to iterative chemistry to improve bioavailability, specificity and potency. The most promising optimized lead compound(s) will then be selected and advanced into preclinical and toxicology studies in preparation for in vivo testing in a murine and1or primate challenge in collaboration with NIAID and USAMRIID investigators. It is anticipated that the results of these experiments will identify one or more antiviral drugs as development candidates to provide a rapid-response defense against the deliberate introduction of a pathogenic poxvirus into the environment. An event which we all hope never transpires, but for which preparation is vital.